大鼠神經痛模式中環導水管灰質區神經可塑性變化之研究

Neuroplasticity changes in the periaqueductal gray in a rat model of neuropathic pain

何昱征(Yu-Cheng Ho)

指導教授 ： 邱麗珠

國立臺灣大學/醫學院/藥理學研究所/博士(2015年)

https://doi.org/10.6342/NTU.2015.00776

摘要

神經痛是一種由於神經性損傷而產生的慢性疼痛。當隨著時間增加，脊髓傳入神經異位性放電降低時，神經痛症狀依舊存在，意味著上脊髓核區功能失調參與神經痛的致病機轉。中腦腹外側環導水管灰質區是一個啟動下行止痛路徑的上脊髓核區，但其在神經痛中所扮演的角色仍然不清楚。因此，本篇論文探討在L5/L6脊神經結紮後誘導的神經痛動物模式下，環導水管灰質區是否會產生神經可塑性變化。本篇論文透過電生理、神經化學、行為等研究法來探討。在脊神經結紮後的2天，大鼠即產生機械性痛覺過敏，此症狀持續超過14天。與對照組做比較，神經結紮後3天與10天的大鼠腦片神經活性表現出：較小的EPSC與延長的神經傳遞反應、較少與較小的mEPSC、較高的PPR、較小的AMPA電流、較小的EPSCAMPA、較大的NMDA電流、較大的EPSCNMDA、較小的EPSCAMPA/NMDA值以及NR1和NR2B蛋白表現量上升，但NR2A、GluR1或GluR2表現量則不變。手術後三天與十天的老鼠腦薄片，其神經可塑性並無顯著差異。總論上述結果，神經結紮會導致環導水管灰質區glutamatergic神經傳導活性降低，這個降低的神經傳遞機制是因突觸前與突觸後共同作用。表現量增加的NMDAR也許貢獻在AMPAR的功能低下，藉由細胞內的重新再分布機制完成。長期glutamatergic神經傳遞功能降低導致環導水管灰質區所投射的下行疼痛抑制訊息降低而最終產生慢性神經性疼痛。
除了發現神經結紮後環導水管灰質區glutamatergic神經傳遞功能降低，本篇論文亦探討此神經傳遞功能降低之細胞機制。神經損傷產生周邊神經的異位性放電以及脊髓興奮性突觸可塑性在神經痛中扮演重要角色，但是腦幹核區對於神經痛之貢獻目前尚未完全了解。因此本篇論文深入探討環導水管灰質區突觸可塑性的變化。在對照組中，adenylyl cyclase(AC)活化劑forskolin可以對環導水管灰質區神經元之EPSC產生長期持續的增益作用。此增益作用是突觸前機制，因forskloin降低PPR、failure rate、增加mEPSE頻率，但不改變mEPSC強度。Forskolin產生的EPSC增益作用可被b-adrenergic致效劑(isoproternol)所模擬、AC抑制劑(SQ22536)、PKA抑制劑(H89)所阻斷，但不受PDE抑制劑(Ro 20-1724)或A1-adenosine拮抗劑(DPCPX)所阻斷。Forskolin和isoproternol所產生的EPSC增益作用在神經結紮組別的老鼠環導水管灰質區腦薄片中是受損的。酵素活性分析在神經結紮老鼠的組別中，發現在突觸小體中的AC活性顯著性降低，但PDE活性並不受影響。除此之外，抑制性神經傳遞在對照組與神經結紮組別並無顯著性差異。動物行為實驗中，在大鼠環導水管灰質區顯微注射forskolin可以顯著性緩解神經結紮所產生的機械性痛覺過敏。總論以上結果，神經結紮會產生異常的疼痛抑制降低，其細胞機制乃起因於突觸前AC活性的降低而使神經傳導物質釋放量減少而降低下行抑制訊息。細胞因AC活性減少而降低環導水管灰質區的神經可塑性，進而對於下行疼痛抑制訊息的減少，而貢獻於長期的神經性疼痛。
在神經結紮後的3-10天，PAG神經元glutamatergic神經傳遞功能降低，而貢獻於神經損傷所產生的神經痛症狀。然而，神經損傷後的glutamatergic神經傳遞功能低下，其病程目前仍然不清楚。因此，本篇論文亦探討在神經結紮後glutamatergic神經傳遞在PAG其病程的進展。本篇結果發現，在神經結紮後的第一天，PAG神經元表現較高頻率的自發性興奮型突觸後電流(sEPSC)，然而不影響sEPSC電流大小。此神經塑性變化並未在GABAergic神經傳遞中發現。SNL1組別中發現降低的PPR，這代表著突觸前的glutamate釋放量可能是增加的。然而，AMPA/NMDA值與細胞興奮性在兩組別中並無顯著性改變。這些結果顯示出，神經結紮後的第一天，在PAG的神經元發現glutamatergic傳遞是功能促進的，而3-10天後則功能轉為低下。這個在神經結紮後的第一天在PAG產生glutamatergic神經傳遞的過渡性興奮或許代表著是一種止痛的保護機制，來對抗因神經損傷而產生的脊髓傳入神經纖維異位性放電。

關鍵字

神經痛 ； 環導水管灰質區 ； 脊髓神經結紮 ； 神經塑性

並列摘要

Neuropathic pain, a chronic pain due to neuronal lesion, remains unaltered even after the injury-induced spinal afferent discharges have declined, suggesting an involvement of supraspinal dysfunction. The midbrain ventrolateral periaqueductal gray (vlPAG) is a crucial supraspinal region for initiating descending pain inhibition while its role in neuropathic pain remains unclear. Therefore, we examined neuroplastic changes in the vlPAG of midbrain slices isolated from neuropathic rats induced by L5/L6 spinal nerve ligation (SNL) via electrophysiological, and neurochemical approaches. Significant mechanical hypersensitivity was induced in rats 2 days after SNL and lasted for more than 14 days. As compared with the sham-operated group, vlPAG slices from neuropathic rats 3 (NP3) and 10 (NP10) days after SNL displayed smaller excitatory post-synaptic currents (EPSCs) with prolonged latency, less frequent and smaller miniature EPSCs, higher paired-pulse ratio of EPSCs, smaller AMPA receptor (AMPAR)-mediated EPSCs (EPSCAMPAs), smaller AMPA currents, greater NMDA receptor (NMDAR)-mediated EPSCs (EPSCNMDAs), greater NMDA currents, lower EPSCAMPA/EPSCNMDA ratios and up-regulation of NR1 and NR2B, but not NR2A, GluR1 or GluR2, subunits of glutamate receptors. There were no significant differences between NP3 and NP10 groups. These results suggest that SNL leads to hypo-glutamatergic neurotransmission in the vlPAG, resulting from both pre-synaptic and post-synaptic mechanisms. Mechanism of up-regulation NMDARs might contribute to hypofunction of AMPARs via subcellular redistribution. Long-term hypo-glutamatergic function in the vlPAG may lead to persistent reduction of descending pain inhibition, resulting in chronic neuropathic pain.
In addition, nerve injury-induced elevation of peripheral neuronal discharge and spinal excitatory synaptic plasticity play an important role in neuropathic pain while little is known about the contribution of changes in the brain stem. Here, we also examined synaptic plasticity changes in the vlPAG, a crucial midbrain region for initiating descending pain inhibition, in SNL-induced neuropathic rats. In vlPAG slices of sham-operated rats, forskolin, an adenylyl cyclase (AC) activator, produced long-lasting enhancement of EPSCs. This is of presynaptic origin since forskolin decreased the paired-pulse ratio and failure rate of EPSCs and increased the frequency, but not amplitude of miniature EPSCs. Forskolin-induced EPSC potentiation was mimicked by a beta-adrenergic agonist (isoproterenol), and prevented by an AC inhibitor (SQ22536) and a cAMP-dependent protein kinase (PKA) inhibitor (H89), but not by a phosphodiesterase (PDE) inhibitor (Ro 20-1724) or an A1-adenosine antagonist (DPCPX). Both forskolin- and isoproterenol-induced EPSC potentiation was impaired in the SNL group. The enzymatic activity of AC, but not PDE, in PAG synaptosomes was significantly decreased after SNL. Interestingly, inhibitory PSCs in vlPAG slices were not different between SNL and sham groups. Intra-vlPAG microinjection of forskolin alleviated SNL-induced mechanical allodynia in rats. These results suggest that SNL leads to inadequate descending pain inhibition due to impaired glutamatergic synaptic plasticity in the PAG, resulting from a deficit in the release machinery mediated by presynaptic AC activity and subsequent cAMP-PKA signaling. This hypofunction of AC-mediated glutamatergic synaptic plasticity in the PAG may contribute to long-term neuropathic pain.
The hypofunction of glutamatergic neurotransmission in the vlPAG of rats 3-10 days after never injury, which may contributes to nerve injury-induced neuropathic pain. However, the progress of this hypo-glutamatergic activity after nerve injury remains unclear. Therefore, we also examined changes in the glutamatergic transmission in vlPAG slices isolated from neuropathic rats 1 day (SNL1) after SNL. We found that, as compared with sham-operated group, the frequency, but not amplitude, of spontaneous excitatory postsynaptic currents (sEPSCs), of vlPAG slices was increased in the SNL1 group. The SNL1 group also had depressed paired-pulse ratio, suggesting glutamate release is increased. However, there was no difference in the EPSCAMPA/NMDA ratio and membrane excitability between sham and SNL1 groups. These results showed that, 1 day after SNL, the glutamatergic neurotransmission was increased in the PAG although it was decreased at 3-10 days. These neuroplastic changes were not found in GABAergic transmission in the PAG of the SNL1 group. This transitional elevation of glutamatergic transmission in the PAG can lead to antinociception and may be an antinociceptive mechanism in the midbrain against peripheral nerve injury induced ectopic discharges in the primary afferents.

並列關鍵字

neuropathic pain ； periaqueductal gray ； spinal nerve ligation ； neuroplasticity

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